Bourbon H M, Martin-Blanco E, Rosen D, Kornberg T B
Department of Biochemistry and Biophysics, University of California, San Francisco 94143, USA.
J Biol Chem. 1995 May 12;270(19):11130-9. doi: 10.1074/jbc.270.19.11130.
The engrailed gene encodes a homeodomain-containing phosphoprotein that binds DNA. Here, we show that engrailed protein is posttranslationally modified in embryos and in embryo-derived cultured cells but is essentially unmodified when expressed in Escherichia coli. Engrailed protein produced by bacteria can be phosphorylated in nuclear extracts prepared from Drosophila embryos, and phosphotryptic peptides from this modified protein partly reproduce two-dimensional maps of phosphotryptic fragments obtained from metabolically labeled engrailed protein. The primary embryonic protein kinase modifying engrailed protein is casein kinase II (CK-II). Analysis of mutant proteins revealed that the in vitro phosphoacceptors are mainly clustered in a region outside the engrailed homeodomain and identified serines 394, 397, 401, and 402 as the targets for CK-II phosphorylation. CK-II-dependent phosphorylation of an N-truncated derivative of engrailed protein purified from bacteria increased its DNA binding 2-4-fold.
engrailed基因编码一种含同源结构域的磷蛋白，该蛋白可结合DNA。在此，我们表明engrailed蛋白在胚胎和胚胎来源的培养细胞中会发生翻译后修饰，但在大肠杆菌中表达时基本未被修饰。细菌产生的engrailed蛋白可在从果蝇胚胎制备的核提取物中被磷酸化，这种修饰蛋白的磷酸化胰蛋白酶肽部分重现了从代谢标记的engrailed蛋白获得的磷酸化胰蛋白酶片段的二维图谱。修饰engrailed蛋白的主要胚胎蛋白激酶是酪蛋白激酶II（CK-II）。对突变蛋白的分析表明，体外磷酸化受体主要聚集在engrailed同源结构域之外的区域，并确定丝氨酸394、397、401和402是CK-II磷酸化的靶点。从细菌中纯化的engrailed蛋白的N端截短衍生物的CK-II依赖性磷酸化使其DNA结合能力提高了2至4倍。
